In this renewal application, we propose to extend our discoveries during the previous funding period on the ion channel protein complex of NALCN, UNC79 and UNC80. We discovered that the NALCN protein forms a complex with UNC79 and UNC80 in mammalian brain and is a major contributor the basal sodium leak conductance in the neurons. The channel is also controlled by neuro-peptides through G protein-coupled receptors but in a G protein-independent fashion. Knocking out Nalcn or Unc79 leads to neonatal lethality and the mutant neurons are less excitable. UNC79 and UNC80 are large novel proteins well conserved among species. They are required for the ion channel function. Despite their large sizes (~3,000 amino acids), they do not have recognizable domains. We will use biochemical and electrophysiological studies to define the interaction domains on each of the proteins, and find out their contribution to the ion channel function (aims 1 and 2). NALCN's G protein-independent activation is quite unique and it provides an opportunity to dissect this unusual ion channel activation pathway by G protein-coupled receptors. In aim 3, we study the mechanisms of this activation by revealing the protein domains and the signaling steps important for the pathway. Results from these studies will help us understand how neuronal excitability is regulated at the molecular level under physiological and pathophysiological conditions such as autism, paralysis, seizure and epilepsy.